1. Field of the Invention
The present invention relates generally to molecular biology and medicine and more specifically to pro-caspases that have been modified to facilitate directed oligomerization, and to methods of using such modified pro-caspases for inducing apoptosis in a cell.
2. Background Information
In essentially all self-renewing tissues, a balance is struck between cell production by mitosis and cell loss due to programmed cell death, thereby maintaining total cell numbers within a physiologically appropriate range. In pathological conditions, however, the balance in cell production and cell loss can be disrupted. In cancer, for example, an increased amount of cell production due to a shortened cell cycle time or to a decreased amount of cell death can occur due to dysregulation of a programmed cell death pathway, resulting in growth of a tumor. Dysregulation of apoptosis also can occur, for example, in neurodegenerative diseases, in which neurons die prematurely, and induction of apoptosis can figure prominently in the pathophysiology of diseases associated with viral infection.
In multicellular organisms, homeostasis is maintained by balancing the rate of ell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular form of programmed cell death, apoptosis, is carried out when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell, for example, in response to genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, and in the absence of an inflammatory response.
Various diseases, including, for example, cancer, inborn errors of metabolism, and neurodegenerative diseases have been refractory to treatment. Recently, however, gene therapy has begun to emerge as a viable means to treat such diseases at the cellular level. Although clinical trials of gene therapy protocols have yet to produce statistically significant results, positive results obtained in patients treated by gene therapy cannot be ignored, particularly since gene therapy generally has been practiced on patients that have failed more conventional treatment protocols.
Although gene therapy holds great promise to alleviate and cure various inherited and acquired diseases, it must be proven not only efficient, but also safe, in order to become a routine clinical procedure. Unfortunately, genetically manipulated cells can acquire unwanted properties and become deleterious to the host due, for example, to insertion of an introduced gene into an otherwise normal gene in the host cell, thus disrupting the function of the normal gene. A method that ablates such cells can provide a safeguard against unwanted deleterious effects and, therefore, is highly desired. Ideally, such a system should effectively and specifically induce apoptosis, a physiologic form of cell death that rids the body of a cell without eliciting a harmful inflammatory response.
The mechanisms that mediate apoptosis have been studied intensively, and involve the activation of endogenous proteases, the loss of mitochondrial function, and the appearance of structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved evolutionarily. However, while numerous genes have been identified as involved in process of apoptosis, the mechanisms by which the products of these genes interact to execute the apoptotic program is not well understood.